Diagnostics and therapeutics for glaucoma

ABSTRACT

Methods and compositions for diagnosing and treating glaucoma are disclosed.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/186,073, filed Feb. 29, 2000, the entire contents of which areincorporated herein by reference.

1. BACKGROUND OF THE INVENTION

[0002] Glaucoma

[0003] Glaucoma is a group of ocular disorders, characterized bydegeneration of the optic nerve. It is one of the leading causes ofblindness worldwide. One major risk factor for developing glaucoma isfamily history. Several different inherited forms of glaucoma have beendescribed.

[0004] Primary congenital or infantile glaucoma is an inherited disorderthat is characterized by an improper development of the aqueous outflowsystem of the eye, which leads to elevated intraocular pressure,enlargement of the glove or cornea (i.e., buphthalmos), damage to theoptic nerve, and eventual visual impairment.

[0005] Primary open angle glaucoma (POAG) is a common disordercharacterized by atrophy of the optic nerve resulting in visual fieldloss and eventual blindness. POAG has been divided into two majorgroups, based on age of onset and differences in clinical presentation.Juvenile-onset POAG usually manifests itself in late childhood or earlyadulthood. Its progression is rapid and severe, with high intraocularpressure. This type of POAG is poorly responsive to medical treatment,and usually requires ocular surgery. Adult- or late-onset POAG is themost common type of glaucoma. It is milder and develops more graduallythan juvenile-onset POAG, with variable onset usually after the age of40. This type of POAG is associated with slight to moderate elevation ofintraocular pressure, and often responds satisfactorily to regularlymonitored medical treatment. Unfortunately, this disease may not bedetected until after irreversible damage to the optic nerve has alreadyoccurred because it progresses gradually and painlessly.

[0006] Both types of POAG are often associated with elevated intraocularpressure as a result of an inhibition of aqueous humor outflow throughthe trabecular meshwork. The pathophysiology of the human trabecularmeshwork (HTM) in POAG has been characterized by an increase inextracellular matrix components and a decrease in the number oftrabecular meshwork cells. It is thus probable that a defect in thestructure, function or number of HTM cells influences the pathogenesisof POAG. The pathophysiology of POAG also involves the cells of thehuman lamina cribrosa (HLC), which has been shown to possess a patternof protein expression that is similar to the HTM (Steely et al. (2000)Exp Eye Res 70: 17-30). Accordingly, POAG may have a common causalorigin in the two tissues most responsible for damage to the neuralretina. Therefore, it will be important to identify and understand thecellular control mechanisms acting within the HTM and the HLC in orderto both understand the molecular etiology of POAG and identify uniquetreatment modalities.

[0007] Cultured HTM cells have been shown to express mRNA for numerousgrowth factor receptors and, furthermore, these expressed receptors havebeen shown to be functional because exogenous growth factoradministration elicits a physiologic response (Wordinger et al. (1998)Invest Ophthalmol Vis Sci 39: 1575-89). In vivo, these receptors may beactivated by growth factors present within the aqueous humor(aquecrine/paracrine) or by growth factors synthesized and releasedlocally by trabecular meshwork cells themselves (autocrine). Indeed,TGF-b isoforms have been shown to significantly inhibit EGF-stimulatedtrabecular meshwork cell proliferation, while FGF-1, TGF-a, EGF, IL-1a,Il-1b, HGF, TNF-a, PDGF-AA, and IGF-1 significantly stimulatedextracellular acidification (ibid.). Specific growth factors actingthrough high-affinity receptors may be involved in maintaining thenormal microenvironment of the HTM and also may be involved in thepathogenesis of POAG.

[0008] One insight into the molecular pathology comes from theobservation that glucocorticoids, which can induce ocular hypertensionin both animals and humans, alter the cytoskeletal structure of culturedHTM cells (Wilson et al. (1993) Current Eye Res 12: 783-93). Thesecytoskeletal changes involve the reorganization of actin microfilamentsinto cross-linked actin networks (CLANs), and this structural alterationmay be the ultimate physiological change which brings about ocularhypertension (Clark et al. (1993) J Glaucoma 4: 183-88). Indeed, thehypotensive steroid tetrahydrocortisol, which has been shown to lowerthe intraocular pressure (IOP) of glucocorticoid-induced ocularhypertension, also appears to inhibit these glucocorticoid-mediatedchanges in the HTM cytoskeleton (Clark et al. (1996) Inv Ophthal & VisSci 37: 805-813).

[0009] U.S. Pat. Nos. 5,925,748, 5,916,778 and 5,885,776 disclosediagnostic methods for glaucoma associated with mutations in the GLC1Agene and assays for identifying glaucoma therapeutics that modulate theactivity of the MYOC protein encoded by the GLC1A gene. The Wntsignaling pathway.

[0010] The Wnt gene family encodes secreted ligand proteins that servekey roles in differentiation and development. This family comprises atleast 15 vertebrate and invertebrate genes including the Drosophilasegment polarity gene wingless and one of its vertebrate homologues,integrated from which the Wnt name derives. The Wnt proteins appear tofacilitate a number of developmental and homeostatic processes. Forexample, vertebrate Wnt1 appears to be active in inducing myotomeformation within the somites and in establishing the boundaries of themidbrain (see McMahon and Bradley (1990) Cell 62: 1073; Ku and Melton(1993) Development 119: 1161; Stern et al. (1995) Development 121:3675). During mammalian gastrulation, Wnt3a, Wnt5a, and Wnt5b areexpressed in distinct yet overlapping regions within the primitivestreak. Wnt3a is the only Wnt protein seen in the regions of the streakthat will generate the dorsal (somite) mesoderm, and mice homozygous fora null allele of the Wnt3a gene have no somites caudal to the forelimbs.The Wnt genes also are important in establishing the polarity ofvertebrate limbs, just as the invertebrate homolog wingless has beenshown to establish polarity during insect limb development. In bothcases there are interactions with Hedgehog family members as well.

[0011] The Wnt signaling pathway comprises a number of proteins involvedin the transduction of Wnt/wingless signaling and is intimatelyconnected to the hedgehog developmental pathway. In Drosophila, thesecreted wingless protein mediates reciprocal interaction between cellsin the wingless-hedgehog pathway by binding to neighboring cells throughthe Frizzled receptor. The Frizzled receptor then activates Dishelveledprotein, which blocks the inhibiting action of Zeste-white-3 kinase uponthe Armadillo protein (a beta-catenin protein). The active Armadilloprotein, acts with the high mobility group (HMG) protein LEF/TCF(Lymphoid Enhancer Factor/T-Cell Factor) to promote nuclear expressionof the hedgehog (hh) gene. Hedgehog is a secreted protein which can bindto cells adjacent to the Wnt/wingless-activated cell through anotherreceptor, the Patched protein. Binding of the Hedgehog protein to thePatched receptor activates nuclear expression of the wingless protein,which is then secreted and further reinforces the reciprocal signalingwith the neighboring hedgehog-secreting cell. The Wnt/Wingless-Hedgehogreciprocal signaling system thereby facilitates the differentialdetermination of two adjacent cells during vertebrate and invertebratedevelopment. This results in the stabilization of a differentiatedborder wherein the tissue on one side secretes Hedgehog protein, whilethe tissue on the other side produces Wingless. Indeed, the cell surfaceplays an extremely critical role in development and homeostasis byeffecting the differential adhesion of one cell to another, as well asto an extracellular matrix. Furthermore, once differential cell adhesionhas occurred, the action of Wnt/Wingless-Hedgehog processes facilitatesthe continued signaling between adjacent cell layers.

[0012] This Wnt/Wingless border is critical in the production ofsegments and appendages in Drosophila as well as brain and limbsubdivisions in the mammals (Ingham (1994) Curr Biol 4: 1; Niswander etal. (1994) Nature 371: 609; Wilder and Perrimon (1995) Development 121:477). In Xenopus, frizzled-2 receptor (xfz2) is highly expressedfollowing gastrulation in the eye anlage and otic vesicle (Deardorff andKlein (1999) Mech Dev 87: 229), and in chicken, a particular Wnt genefamily member, Wnt13, has been shown to be expressed in theproliferative epithelium of the lens and both pigmented andnon-pigmented layers of the ciliary margin (Jasoni et al. (1999) Dev Dyn215: 215). The reciprocal Wnt/Wingless-Hedgehog pathway may also play arole in the maintenance of normal differentiated somatic tissue. Forexample, in human, sporadic loss-of-function mutations of the patchedgene in somatic tissues causes basal cell carcinomas, the most commontype of human cancer. Furthermore, heritable mutations of the patchedgene give rise to basal cell nevus syndrome, an autosomal dominantcondition characterized by developmental abnormalities, including riband craniofacial alterations, and malignant tumors (Hahn et al. (1996)Cell 85: 841; Johnson et al. (1996) Science 272: 1668).

[0013] Recently a protein homologous to mammalian Wnt receptor Frizzled,termed the secreted or soluble frizzled related protein 5 (SFRP5) hasbeen shown to be preferentially expressed by the vertebrate retinalpigment epithelium (RPE) (Chang et al. (1999) Hum Mol Genet 8: 575).Furthermore, another SFRP, SPRP2 has been shown to be expressedspecifically by cells of the inner nuclear layer. As a result,photoreceptor cells of the retina are exposed to two opposing gradientsof SFRP molecules. Because the frizzled related proteins do not containa membrane spanning domain, they are thought to be a secreted, solubleform of the receptor which interferes with Wnt signaling through thenormal seven transmembrane Frizzled receptor. Indeed, FrzA, an sFRP thatis highly expressed in vascular endothelium and a variety of epithelium,specifically binds to Wnt-1 protein and thereby blocks Wnt-1 signalingthrough the Frizzled receptor (Dennis et al. (1999) J Cell Sci 112:3815).

2. SUMMARY OF THE INVENTION

[0014] In one aspect, the present invention provides novel methods andkits for determining whether a subject has or is predisposed todeveloping glaucoma. In one embodiment, the method is based ondetermining the relative level or activity of a Frizzle Related Protein(FRP), a wingless (Wnt) signaling pathway component, a gene activated byWnt signaling or the gene product of a gene activated by Wnt signaling.In preferred embodiments, the assay is performed on trabecular meshworkcells obtained from a subject. The method can include detecting inappropriate cells, the presence or absence of a genetic lesioncharacterized by at least one of: (i) a mutation of a gene encodingFrizzle Related Protein (FRP-1), a Wnt signaling component or a genewhose expression is activated by Wnt signaling; (ii) the misexpressionof FRP, a Wnt signaling component or a gene whose expression isactivated by Wnt signaling; or (iii) an error or mutation in thepromoter regulating FRP, a Wnt signaling component or a gene whoseexpression is activated by Wnt signaling, said error or mutation leadingto aberrant expression.

[0015] In particularly preferred embodiments, the diagnostic methodscomprise ascertaining the existence of at least one of (a) a deletion ofone or more nucleotides from a wildtype FRP, Wnt signaling component ora gene whose expression is activated by Wnt signaling; (b) an additionof one or more nucleotides to a wildtype FRP, Wnt signaling component ora gene whose expression is activated by Wnt signaling; (c) asubstitution of one or more nucleotides of a wildtype FRP, Wnt signalingcomponent or a gene whose expression is activated by Wnt signaling; (d)a gross chromosomal rearrangement of a wildtype FRP, Wnt signalingcomponent or a gene whose expression is activated by Wnt signaling; (e)an alteration in the level of a messenger RNA (mRNA) transcript of aFRP, Wnt signaling component or a gene whose expression is activated byWnt signaling; (f) the presence of a non-wildtype splicing pattern of amRNA transcript of an FRP, Wnt signaling component or a gene whoseexpression is activated by Wnt signaling; (g) an aberrant level oractivity of an FRP, Wnt signaling protein or a protein encoded by a genewhose expression is activated by Wnt signaling.

[0016] For example, a genetic lesion can be detected by: (i) providingprobes and primers comprised of an oligonucleotide, which hybridizes toa sense or antisense sequence of an FRP, Wnt signaling component or agene whose expression is activated by Wnt signaling (wildtype or mutant)or fragment thereof or 5′ or 3′ flanking sequence naturally associatedwith an FRP, Wnt signaling component or a gene whose expression isactivated by Wnt signaling; (ii) contacting the probes or primers to anappropriate nucleic acid containing biological sample obtained from thesubject; and (iii) detecting, by hybridization of the probes or primersto the nucleic acid, the presence or absence of the genetic lesion. In apreferred embodiment, the diagnostic methods and/or kits utilize a setof primers for amplifying (e.g. via PCR or LCR) at least one region ofan FRP, Wnt signaling component or a gene whose expression is activatedby Wnt signaling that may contain a mutation, and means for analyzingthe amplification product for differences mutations or gene expressionlevels from the normal, wildtype coding sequence. In another preferredembodiment, the diagnostic methods and/or kits utilize a probe todetermine its ability to hybridize under appropriately stringentconditions to a complementary nucleic acid sequence in the biologicalsample, wherein an inability of a probe, which is comprised of awildtype FRP, Wnt signaling component or a gene whose expression isactivated by Wnt signaling, to hybridize to the sample nucleic acid isindicative of the presence of a mutation in the sample nucleic acid; orthe ability of a probe which is comprised of a mutant FRP, Wnt signalingcomponent or a gene whose expression is activated by Wnt signaling, tohybridize to the sample nucleic acid is indicative of the presence of amutation in the sample nucleic acid. In another preferred embodiment,the protein level or activity of an FRP, Wnt signaling component or aprotein encoded by a gene whose expression is activated by Wnt signalingcan be detected using any of a variety of methods, includingimmunodetection and biochemical tests.

[0017] Information obtained using the assays and kits described herein(alone or in conjunction with information on another genetic defect orenvironmental factor, which contributes to the development of glaucoma)is useful for determining whether a non-symptomatic subject has or islikely to develop glaucoma. In addition, the information can allow amore customized approach to the prevention or treatment of the disorder.

[0018] In another aspect, the invention provides in vitro or in vivoassays for screening test compounds to identify therapeutics fortreating or preventing glaucoma. In particularly preferred embodiments,the therapeutics promote Wnt signaling. In one embodiment, the method isa binding assay, which consists essentially of the steps of (a) forminga reaction mixture, including: (i) an FRP or Wnt signaling polypeptide,(ii) an FRP or Wnt signaling polypeptide binding partner, and (iii) atest compound and (b) detecting interaction of the FRP or Wnt signalingpolypeptide and the binding protein. A statistically significant change(potentiation or inhibition) in the interaction of the FRP or Wntsignaling polypeptide and the binding protein in the presence of thecompound, indicates a potential agonist (mimetic or potentiator) orantagonist (inhibitor) of Wnt signaling. The reaction mixture can be acell-free protein preparation, e.g. a reconstituted protein mixture or acell lysate, a cultured cell system, or it can be a recombinant cellincluding a heterologous nucleic acid recombinantly expressing thebinding partner.

[0019] Yet another exemplary embodiment provides an assay for screeningtest compounds to identify agents which promote or increase the rate ofWnt signaling and/or expression of genes, which are regulated by Wntsignaling in the trabecular meshwork. In one embodiment, the screeningassay comprises contacting a cell transfected with a reporter geneoperably linked to a promoter, which is regulated by a high mobilitygroup (HMG) protein (e.g. Lymphoid Enhancer Factor/T-Cell Factor) with atest compound and determining the level of expression of the reportergene. The reporter gene can encode, e.g., a gene product that gives riseto a detectable signal such as: color, fluorescence, luminescence, cellviability, relief of a cell nutritional requirement, cell growth, anddrug resistance. For example, the reporter gene can encode a geneproduct selected from the group consisting of chloramphenicol acetyltransferase, luciferase, beta-galactosidase and alkaline phosphatase.

[0020] In a further aspect, the invention features methods for treatingglaucoma by contacting appropriate cells (e.g. trabecular meshworkcells) with an effective amount of a compound that promotes theexpression of trabecular meshwork genes that are involved in or areregulated by Wnt signaling. Preferred compounds can be small molecules,nucleic acids (including antisense or triplex molecules and ribozymes),proteins, peptides or peptide mimetics. Particularly preferred compoundsare Frizzle Related Protein (FRP) antagonists. Particularly preferredantagonists are antisense, ribozyme or triplex molecules that inhibit ordecrease the level of FRP expressed in cells. Other preferred FRPantagonists are antibodies, which reduce or inhibit FRP binding to Wnt.

[0021] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

3. BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 shows the cDNA sequence of the human Frizzle RelatedProtein (FRP-1) (Seq ID No. 1).

[0023]FIG. 2 shows the amino acid sequence of the human FRP-1.

[0024]FIG. 3 schematically depicts the Wnt signal transduction pathway.

[0025]FIG. 3(a) shows the inhibition of gene expression based on bindingof FRP to Wnt.

[0026]FIG. 3(b) shows Wnt stimulated gene expression. The binding of Wntto a frizzled protein (Fz) activates disheveled (Dsh) which in turnprevents the binding of glycogen-synthase-kinase 3 (GSK3) to proteinkinase C (APC), which results in the accumulation of β-catenin, which inturn facilitates interactions with the transcription factor, T cellfactor (TCF), promoting gene expression.

4. DETAILED DESCRIPTION OF THE INVENTION

[0027] 4.1. General

[0028] In general, the invention is based on the finding that frizzledrelated protein-1 (FRP-1) is upregulated in the trabecular meshwork (TM)of glaucoma patients. Although not wishing to be bound, it is thoughtthat the Wnt signaling pathway works in the TM as depicted in FIG. 3(b)to regulate important trabecular meshwork cell functions and that FRP-1antagonizes normal Wnt signaling, as shown in FIG. 3(a), therebyinterfering with TM cell function.

[0029] 4.2 Definitions

[0030] For convenience, the meaning of certain terms and phrasesemployed in the specification, examples, and appended claims areprovided below.

[0031] The term “aberrant”, as used herein is meant to refer to analteration in a gene product level or bioactivity which is found in aglaucomatous tissue or cells but not in a nonglaucomatous tissue orcells. For example, an aberrantly high level of frizzle related proteingene product is associated with glaucomatous trabecular meshwork cellsobtained from a glaucoma patient than from nonglaucomatous trabecularmeshwork cells obtained from a normal patient. Furthermore, anaberrantly low bioactivity of Wnt pathway components is associated withtrabecular meshwork cells from a normal patient.

[0032] The term “an aberrant activity”, as applied to an activity of apolypeptide such as FRP, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart. Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide. A cell can have an aberrant FRP activity due to overexpressionor underexpression of the gene encoding FRP.

[0033] The term “agonist”, as used herein, is meant to refer to an agentthat directly or indirectly enhances, supplements or potentiates Wntinitiated gene expression or the level or activity of a protein encodedby a Wnt regulated gene or a gene or protein in the Wnt signalingpathway.

[0034] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for the gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions and insertions of nucleotides. Anallele of a gene can also be a form of a gene containing a mutation.

[0035] The term “antagonist”, as used herein, is refers to an agent thatdirectly or indirectly prevents, minimizes or suppresses Wnt initiatedgene expression or the level or activity of a protein encoded by a Wntregulated gene or a gene or protein in the Wnt signaling pathway.

[0036] The term “binding partner”, as used herein refers to acomposition of matter that interacts though noncolavent forces with aspecified gene product. For example, “binding partners” of the frizzledrelated protein gene product include compositions of matter whichinteract with frizzled related protein gene mRNAs, such as an FRP-1antisense polynucleotide, and compositions which interact with frizzledrelated protein polypeptides, such as Wnt polypeptides.

[0037] As used herein the term “bioactive fragment of a polypeptide”refers to a fragment of a full-length polypeptide, wherein the fragmentspecifically mimics or antagonizes an activity of the correspondingfull-length wild-type polypeptide.

[0038] “Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0039] A “chimeric polypeptide” or “fusion polypeptide” is a fusion of afirst amino acid sequence encoding, for example, one of the subject FRPpolypeptides with a second amino acid sequence defining a domain (e.g.polypeptide portion) foreign to and not substantially homologous withany domain of an FRP polypeptide. A chimeric polypeptide may present aforeign domain which is found (albeit in a different polypeptide) in anorganism which also expresses the first polypeptide, or it may be an“interspecies”, “intergenic”, etc. fusion of polypeptide structuresexpressed by different kinds of organisms. In general, a fusionpolypeptide can be represented by the general formula X-FRP-Y, whereinFRP represents a portion of the polypeptide which is derived from an FRPpolypeptide, and X and Y are independently absent or represent aminoacid sequences which are not related to an FRP sequence in an organism,including naturally occurring mutants.

[0040] A “delivery complex” shall mean a targeting means (e.g. amolecule that results in higher affinity binding of a gene, protein,polypeptide or peptide to a target cell surface and/or increasedcellular or nuclear uptake by a target cell). Examples of targetingmeans include: sterols (e.g. cholesterol), lipids (e.g. a cationiclipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associatedvirus, and retrovirus) or target cell specific binding agents (e.g.ligands recognized by target cell specific receptors). Preferredcomplexes are sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex is cleavable under appropriate conditions within the cell sothat the gene, protein, polypeptide or peptide is released in afunctional form.

[0041] As is well known, genes may exist in single or multiple copieswithin the genome of an individual. Such duplicate genes may beidentical or may have certain modifications, including nucleotidesubstitutions, additions, inversions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding an FRP polypeptide” may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a polypeptide with the same biological activity.

[0042] A “Frizzled Related Protein (FRP)” can be any member of a familyof secreted proteins with similarity to the extracellular,ligand-binding domain of Frizzled proteins. FRPs are also referred to assecreted or soluble frizzled-related protein (sFRPs) because they do notcontain a membrane spanning domain and hence appear to act asdominant-negative receptors of Wnt proteins. FRPs are encoded by anumber of different vertebrate genes including: the human secretedfrizzled-related protein encoding gene Frz-1 (GenBank Accession No.AF056087); the human secreted frizzled-related protein encoding geneSFRP5 (GenBank Accession No. AF117758); the human FrzB gene (GenBankAccession No. U24163); and the Xenopus FrzA gene (Genbank Accession No.AF049908).

[0043] The term “glaucoma”, as used herein refers to a group of eyediseases characterized by characteristic degeneration of the optic nervehead and visual field loss, which is often caused by increasedintraocular pressure due to blockage of the channel through whichaqueous humor drains (chronic or open-angle glaucoma) or by pressure ofthe iris against the lens (acute or angle-closure glaucoma).

[0044] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are identical at that position. A degree of homology orsimilarity or identity between nucleic acid sequences is a function ofthe number of identical or matching nucleotides at positions shared bythe nucleic acid sequences. A degree of identity of amino acid sequencesis a function of the number of identical amino acids at positions sharedby the amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.The percentage homology between two nucleic acid or polypeptide sequencecan be determined using any of several mathematical algorithms which arewell known in the art (as provided, for example, by the BLAST sequencehomology software available at: http://www.ncbi.nlm.nih.gov/BLAST/). An“unrelated” or “non-homologous” sequence shares less than 40% identity,though preferably less than 25% identity, with one of the FRP sequencesof the present invention.

[0045] The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may, forexample, be protein-protein or protein-nucleic acid or nucleicacid-nucleic acid in nature.

[0046] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject FRP polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks the FRPgene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

[0047] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation (e.g., by agonizing or potentiating))and downregulation (i.e. inhibition or suppression (e.g., byantagonizing, decreasing or inhibiting)).

[0048] The term “mutated gene” refers to an allelic form of a gene,which is capable of altering the phenotype of a subject having themutated gene relative to a subject which does not have the mutated gene.If a subject must be homozygous for this mutation to have an alteredphenotype, the mutation is said to be recessive. If one copy of themutated gene is sufficient to alter the phenotype of the subject, themutation is said to be dominant. If a subject has one copy of themutated gene and has a phenotype that is intermediate between that of ahomozygous and that of a heterozygous subject (for that gene), themutation is said to be co-dominant.

[0049] The “non-human animals” of the invention include mammals such asrodents, non-human primates, sheep, dogs, cows, chickens, amphibians,reptiles, rabbits, etc. Preferred non-human animals are selected fromthe rodent family including rat and mouse, most preferably mouse, thoughtransgenic amphibians, such as members of the Xenopus genus, andtransgenic chickens can also provide important tools for understandingand identifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant genes is present and/or expressed or disrupted in sometissues but not others.

[0050] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0051] The term “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID NO. x” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having SEQ ID NO. x.The term “complementary strand” is used herein interchangeably with theterm “complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID NO. x refers to the complementary strand ofthe strand having SEQ ID NO. x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID NO. x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID NO. x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID NO. x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction.

[0052] The term “polymorphism” refers to the coexistence of more thanone form of a gene or portion (e.g., allelic variant) thereof. A portionof a gene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

[0053] A “polymorphic gene” refers to a gene having at least onepolymorphic region.

[0054] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0055] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to an amino acid-containing geneproduct.

[0056] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding an FRP polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant FRP gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native FRP polypeptide, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the polypeptide.

[0057] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention to identifycompounds that modulate FRP or Wnt signaling bioactivities.

[0058] As used herein, the term “specifically hybridizes” or“specifically detects” refers to the ability of a nucleic acid moleculeof the invention to hybridize to at least approximately 6, 12, 20, 30,50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of avertebrate, preferably an FRP gene.

[0059] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked.

[0060] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of an FRP polypeptide or,in the case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the FRP polypeptide isdisrupted.

[0061] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the FRP polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, is homologousto an endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

[0062] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA.

[0063] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0064] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and expression of nucleic acids to which they arelinked. Vectors capable of directing the expression of genes to whichthey are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

[0065] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0066] A “Wnt signaling component” refers to a protein or gene encodinga protein involved in a Wnt signaling pathway. Examples of such proteinsinclude: Wnt, frizzled (Fz), disheveled (Dsh), glycogen synthase kinase3 (GSK3), protein kinase C (APC), β-catenins, and high mobility group(HMG) proteins (e.g. LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor)).

[0067] “Wnt protein” refers to is encoded by a large group of mammaliangenes including Wnt3a, Wnt5a, and Wnt5b.

[0068] 4.3. Prognostics and Diagnostics for Glaucoma

[0069] Based on the instant disclosed finding that certain subjects withglaucoma have increased levels of FRP, a variety of glaucoma diagnosticscan be developed. Certain diagnostics can detect mutations in nucleicacid sequences that result in inappropriately high levels of FRP. Thesediagnostics can be developed based on the known nucleic acid sequence ofhuman FRP cDNA, as shown in FIG. 1 or the encoded amino acid sequence,which is shown in FIG. 2. Other diagnostics can be developed based onthe genomic sequence of human FRP or of the sequence of genes thatregulate FRP expression. Still other diagnostics can be developed basedupon a change in the level of FRP gene expression at the mRNA level. Aplasmid containing the genomic sequence of human FRP was deposited withthe American Type Culture Collection on ______ and has been assignedATCC Designation No. ______.

[0070] Other diagnostics can detect the activity or level of Wntsignaling proteins or genes encoding Wnt signaling proteins. Forexample, diagnostics can be developed that detect inappropriately lowWnt signaling activity, including for example, mutations that result ininappropriate functioning of Wnt signaling components, including:frizzled (Fz); disheveled (Dsh); glycogen synthase kinase 3 (GSK3),protein kinase C (APC), β-catenins high mobility group (HMG) proteins(e.g. LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor)), and hedgehog(Hh). In addition, non-nucleic acid based techniques may be used todetect alteration in the amount or specific activity of any of these Wntsignaling proteins.

[0071] A variety of means are currently available for detecting aberrantlevels or activities of genes and gene products. For example, manymethods are available for detecting specific alleles at humanpolymorphic loci. The preferred method for detecting a specificpolymorphic allele will depend, in part, upon the molecular nature ofthe polymorphism. For example, the various allelic forms of thepolymorphic locus may differ by a single base-pair of the DNA. Suchsingle nucleotide polymorphisms (or SNPs) are major contributors togenetic variation, comprising some 80% of all known polymorphisms, andtheir density in the human genome is estimated to be on average 1 per1,000 base pairs. SNPs are most frequently biallelic-occurring in onlytwo different forms (although up to four different forms of an SNP,corresponding to the four different nucleotide bases occurring in DNA,are theoretically possible). Nevertheless, SNPs are mutationally morestable than other polymorphisms, making them suitable for associationstudies in which linkage disequilibrium between markers and an unknownvariant is used to map disease-causing mutations. In addition, becauseSNPs typically have only two alleles, they can be genotyped by a simpleplus/minus assay rather than a length measurement, making them moreamenable to automation.

[0072] A variety of methods are available for detecting the presence ofa particular single nucleotide polymorphic allele in an individual.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

[0073] Several methods have been developed to facilitate analysis ofsingle nucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No.4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

[0074] In another embodiment of the invention, a solution-based methodis used for determining the identity of the nucleotide of a polymorphicsite. Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

[0075] An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

[0076] Recently, several primer-guided nucleotide incorporationprocedures for assaying polymorphic sites in DNA have been described(Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov,B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A. -C., et al.,Genomics 8:684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad.Sci. (U.S.A.) 88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. etal., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA™in that they all rely on the incorporation of labeled deoxynucleotidesto discriminate between bases at a polymorphic site. In such a format,since the signal is proportional to the number of deoxynucleotidesincorporated, polymorphisms that occur in runs of the same nucleotidecan result in signals that are proportional to the length of the run(Syvanen, A. -C., et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0077] For mutations that produce premature termination of proteintranslation, the protein truncation test (PTT) offers an efficientdiagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21;van der Luijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA isinitially isolated from available tissue and reverse-transcribed, andthe segment of interest is amplified by PCR. The products of reversetranscription PCR are then used as a template for nested PCRamplification with a primer that contains an RNA polymerase promoter anda sequence for initiating eukaryotic translation. After amplification ofthe region of interest, the unique motifs incorporated into the primerpermit sequential in vitro transcription and translation of the PCRproducts. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresisof translation products, the appearance of truncated polypeptidessignals the presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

[0078] Any cell type or tissue may be utilized to obtain nucleic acidsamples for use in the diagnostics described herein. In a preferredembodiment, the DNA sample is obtained from a bodily fluid, e.g., blood,obtained by known techniques (e.g. venipuncture) or saliva.Alternatively, nucleic acid tests can be performed on dry samples (e.g.hair or skin).

[0079] Diagnostic procedures may also be performed in situ directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, NY).

[0080] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, profiles may also be assessed in suchdetection schemes. Fingerprint profiles may be generated, for example,by utilizing a differential display procedure, Northern analysis and/orRT-PCR.

[0081] A preferred detection method is allele specific hybridizationusing probes overlapping a region of at least one allele of a Wntsignaling component that is indicative of glaucoma and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.In a preferred embodiment of the invention, several probes capable ofhybridizing specifically to other allelic variants involved in glaucomaare attached to a solid phase support, e.g., a “chip” (which can hold upto about 250,000 oligonucleotides). Oligonucleotides can be bound to asolid support by a variety of processes, including lithography. Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

[0082] These techniques may also comprise the step of amplifying thenucleic acid before analysis. Amplification techniques are known tothose of skill in the art and include, but are not limited to cloning,polymerase chain reaction (PCR), polymerase chain reaction of specificalleles (ASA), ligase chain reaction (LCR), nested polymerase chainreaction, self sustained sequence replication (Guatelli, J. C. et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci.USA 86:1173-1177), and Q-Beta Replicase (Lizardi, P. M. et al., 1988,Bio/Technology 6:1197).

[0083] Amplification products may be assayed in a variety of ways,including size analysis, restriction digestion followed by sizeanalysis, detecting specific tagged oligonucleotide primers in thereaction products, allele-specific oligonucleotide (ASO) hybridization,allele specific 5′ exonuclease detection, sequencing, hybridization, andthe like.

[0084] PCR based detection means can include multiplex amplification ofa plurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

[0085] In a merely illustrative embodiment, the method includes thesteps of (i) collecting a sample of cells from a patient, (ii) isolatingnucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,(iii) contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one allele of a Wntsignaling component that is indicative of glaucoma under conditions suchthat hybridization and amplification of the allele occurs, and (iv)detecting the amplification product. These detection schemes areespecially useful for the detection of nucleic acid molecules if suchmolecules are present in very low numbers.

[0086] In a preferred embodiment of the subject assay, aberrant levelsor activities of Wnt signaling components that are indicative ofglaucoma are identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis.

[0087] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the allele.Exemplary sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert ((1977) Proc. Natl Acad Sci USA 74:560)or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci USA 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures may be utilized when performing the subject assays (see, forexample Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example PCT publication WO 94/16101; Cohen et al.(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159). It will be evident to one of skill in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-track or the like, e.g., where only one nucleic acid isdetected, can be carried out.

[0088] In a further embodiment, protection from cleavage agents (such asa nuclease, hydroxylamine or osmium tetraoxide and with piperidine) canbe used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; andSaleeba et al (1992) Methods Enzymol. 217:286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

[0089] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes). Forexample, the mutY enzyme of E. coli cleaves A at G/A mismatches and thethymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches(Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to anexemplary embodiment, an appropriate probe is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

[0090] In other embodiments, alterations in electrophoretic mobilitywill be used to identify aberrant levels or activities of Wnt signalingcomponents that are indicative of glaucoma. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control locusalleles are denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0091] In yet another embodiment, the movement of alleles inpolyacrylamide gels containing a gradient of denaturant is assayed usingdenaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

[0092] Examples of other techniques for detecting alleles include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labeledtarget DNA.

[0093] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage-based detection (Gasparini et al (1992) Mol.Cell Probes 6: 1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

[0094] In another embodiment, identification of an allelic variant iscarried out using an oligonucleotide ligation assay (OLA), as described,e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988)Science 241:1077-1080). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

[0095] Several techniques based on this OLA method have been developedand can be used to detect aberrant levels or activities of Wnt signalingcomponents that are indicative of glaucoma. For example, U.S. Pat. No.5,593,826 discloses an OLA using an oligonucleotide having 3′-aminogroup and a 5′-phosphorylated oligonucleotide to form a conjugate havinga phosphoramidate linkage. In another variation of OLA described in Tobeet al. ((1996) Nucleic Acids Res 24: 3728), OLA combined with PCRpermits typing of two alleles in a single microliter well. By markingeach of the allele-specific primers with a unique hapten, i.e.digoxigenin and fluorescein, each OLA reaction can be detected by usinghapten specific antibodies that are labeled with different enzymereporters, alkaline phosphatase or horseradish peroxidase. This systempermits the detection of the two alleles using a high throughput formatthat leads to the production of two different colors.

[0096] Another embodiment of the invention is directed to kits fordetecting a predisposition for developing glaucoma. This kit may containone or more oligonucleotides, including 5′ and 3′ oligonucleotides thathybridize 5′ and 3′ to at least one Wnt signaling component. PCRamplification oligonucleotides should hybridize between 25 and 2500 basepairs apart, preferably between about 100 and about 500 bases apart, inorder to produce a PCR product of convenient size for subsequentanalysis.

[0097] For use in a kit, oligonucleotides may be any of a variety ofnatural and/or synthetic compositions such as syntheticoligonucleotides, restriction fragments, cDNAs, synthetic peptidenucleic acids (PNAs), and the like. The assay kit and method may alsoemploy labeled oligonucleotides to allow ease of identification in theassays. Examples of labels which may be employed include radio-labels,enzymes, fluorescent compounds, streptavidin, avidin, biotin, magneticmoieties, metal binding moieties, antigen or antibody moieties, and thelike.

[0098] The kit may, optionally, also include DNA sampling means. DNAsampling means are well known to one of skill in the art and caninclude, but not be limited to substrates, such as filter papers, andthe like; DNA purification reagents such as Nucleon™ kits, lysisbuffers, proteinase solutions and the like; PCR reagents, such as 10×reaction buffers, thermostable polymerase, dNTPs, and the like; andallele detection means such as restriction enzyme, allele specificoligonucleotides, degenerate oligonucleotide primers for nested PCR fromdried blood.

[0099] 4.4. Screening Assays for Glaucoma Therapeutics

[0100] The invention further provides screening methods for identifyingglaucoma therapeutics. A glaucoma therapeutic can be any type ofcompound, including a protein, a peptide, peptidomimetic, smallmolecule, and nucleic acid. A nucleic acid can be, e.g., a gene, anantisense nucleic acid, a ribozyme, or a triplex molecule. A glaucomatherapeutic of the invention can be an agonist of a Wnt signalingcomponent activity or an antagonist of FRP or a Wnt signalingantagonistic activity. Preferred agonists include Wnt signalingcomponents or genes and proteins whose expression is regulated by Wntsignaling.

[0101] The invention also provides screening methods for identifyingglaucoma therapeutics which are capable of binding to an FRP protein,thereby interfering with its blocking of Wnt signaling or therapeutics,which are capable of binding to a Wnt signaling component, therebyagonizing the Wnt signaling component activity.

[0102] The compounds of the invention can be identified using variousassays depending on the type of compound and activity of the compoundthat is desired. Set forth below are at least some assays that can beused for identifying glaucoma therapeutics. It is within the skill ofthe art to design additional assays for identifying glaucomatherapeutics based on the Wnt signaling based activation of trabecularmeshwork genes.

[0103] 4.4.1 Cell-free Assays

[0104] Cell-free assays can be used to identify compounds which arecapable of interacting with an FRP, Wnt signaling component or a bindingpartner thereof. Such a compound can, e.g., modify the structure of anFRP, Wnt signaling component or binding partner and thereby effect itsactivity. Cell-free assays can also be used to identify compounds whichmodulate the interaction between an FRP or Wnt signaling component andan binding partner. In a preferred embodiment, cell-free assays foridentifying such compounds consist essentially in a reaction mixturecontaining an FRP or Wnt signaling component and a test compound or alibrary of test compounds in the presence or absence of a bindingpartner. A test compound can be, e.g., a derivative of a bindingpartner, e.g., an biologically inactive target peptide, or a smallmolecule.

[0105] Accordingly, one exemplary screening assay of the presentinvention includes the steps of contacting an FRP, Wnt signalingcomponent or functional fragment thereof or a binding partner with atest compound or library of test compounds and detecting the formationof complexes. For detection purposes, the molecule can be labeled with aspecific marker and the test compound or library of test compoundslabeled with a different marker. Interaction of a test compound with anFRP, Wnt signaling component or fragment thereof or binding partnerthereof can then be detected by determining the level of the two labelsafter an incubation step and a washing step. The presence of two labelsafter the washing step is indicative of an interaction.

[0106] An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the FRP, Wnt signaling component,functional fragment thereof, or binding partner thereof is then flowncontinuously over the sensor surface. A change in the resonance angle asshown on a signal recording, indicates that an interaction has occurred.This technique is further described, e.g., in BIA technology Handbook byPharmacia.

[0107] Another exemplary screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) anFRP or Wnt signaling component, (ii) a binding partner thereof; and(iii) a test compound; and (b) detecting interaction of the FRP or Wntsignaling component and the binding protein. The FRP or Wnt signalingcomponent and binding partner can be produced recombinantly, purifiedfrom a source, e.g., plasma, or chemically synthesized, as describedherein. A statistically significant change (potentiation or inhibition)in the interaction of the FRP or Wnt signaling component and the bindingprotein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of FRP or Wntsignaling bioactivity for the test compound. The compounds of this assaycan be contacted simultaneously. Alternatively, an FRP or Wnt signalingcomponent can first be contacted with a test compound for an appropriateamount of time, following which the binding partner is added to thereaction mixture. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. In the control assay,isolated and purified FRP or Wnt signaling components are added to acomposition containing the FRP binding partner or Wnt signalingcomponent binding partner, and the formation of a complex is quantitatedin the absence of the test compound.

[0108] Complex formation between an FRP protein and an FRP bindingpartner may be detected by a variety of techniques. Modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled, fluorescently labeled, orenzymatically labeled FRP, Wnt signaling component or binding partners,by immunoassay, or by chromatographic detection.

[0109] Typically, it will be desirable to immobilize FRP, a Wntsignaling component or its binding partner to facilitate separation ofcomplexes from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of FRP or a Wntsignaling component to a binding partner, can be accomplished in anyvessel suitable for containing the reactants. Examples includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the protein to be bound to a matrix. For example,glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe binding partner, e.g. a 35S-labeled binding partner, and the testcompound, and the mixture incubated under conditions conducive tocomplex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintilant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level of theFRP or Wnt signaling component or binding partner found in the beadfraction quantitated from the gel using standard electrophoretictechniques such as described in the appended examples.

[0110] Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, FRP, a Wntsignaling component or its cognate binding partner can be immobilizedutilizing conjugation of biotin and streptavidin. For instance,biotinylated FRP or Wnt signaling components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well known in theart (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with FRP or a Wntsignaling component can be derivatized to the wells of the plate, andFRP or Wnt signaling components trapped in the wells by antibodyconjugation. As above, preparations of an FRP or Wnt signalingcomponent, a binding protein and a test compound are incubated in theFRP or Wnt signaling component presenting wells of the plate, and theamount of complex trapped in the well can be quantitated. Exemplarymethods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the FRP or Wnt signalingcomponent binding partner, or which are reactive with FRP or a Wntsignaling component protein and compete with the binding partner; aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the binding partner, either intrinsic orextrinsic activity. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with the bindingpartner. To illustrate, the binding partner can be chemicallycross-linked or genetically fused with horseradish peroxidase, and theamount of polypeptide trapped in the complex can be assessed with achromogenic substrate of the enzyme, e.g. 3,3′-diaminobenzadineterahydrochloride or 4-chloro-1-napthol. Likewise, a fusion proteincomprising the polypeptide and glutathione-S-transferase can beprovided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J BiolChem 249:7130).

[0111] For processes which rely on immunodetection for quantitating oneof the proteins trapped in the complex, antibodies against the proteincan be used. Alternatively, the protein to be detected in the complexcan be “epitope tagged” in the form of a fusion protein which includes,in addition to the FRP or Wnt signaling component sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, N.J.).

[0112] Cell-free assays can also be used to identify compounds whichinteract with an FRP or Wnt signaling component and modulate theiractivity. Accordingly, in one embodiment, an FRP or Wnt signalingcomponent is contacted with a test compound and the catalytic activityof FRP or the Wnt signaling component is monitored. In one embodiment,the ability of FRP or a Wnt signaling component to bind to a targetpeptide is determined according to methods known in the art.

[0113] 4.4.2. Cell Based Assays

[0114] In addition to cell-free assays, such as described above, FRPproteins as provided by the present invention, facilitate the generationof cell-based assays, e.g., for identifying small molecule agonists orantagonists. In one embodiment, a cell expressing an FRP protein on theouter surface of its cellular membrane is incubated in the presence of atest compound alone or a test compound and a molecule which is known tointeract with FRP and the interaction between FRP and a test compound isdetected, e.g., by using a microphysiometer (McConnell et al. (1992)Science 257:1906). An interaction between the FRP protein the testcompound is detected by the microphysiometer as a change in theacidification of the medium. In preferred embodiments, the cell basedassays of the invention utilize human cells obtained from the trabecularmeshwork ocular tissue of normal or glaucoma-affected patients.

[0115] The propagation of human trabecular cells in culture allows thestudy of the structural and functional properties of this distinct celltype under reproducible experimental conditions. Human trabecular cellscan be effectively grown from dissected explants of trabecular tissue,and the cultured cells can maintain the distinctive ultrastructuralfeatures of uncultured trabecular cells through numerous passages invitro. The trabecular cell possesses a wide range of biochemical andstructural properties that may be important for the maintenance of theaqueous outflowpathway. These properties include the growth oftrabecular cells as an endothelial monolayer with a nonthrombogenic cellsurface, the production of plasminogen activator, avid phagocytosis, andthe ability to synthesize glycosaminoglycans, collagen, fibronectin, andother connective tissue elements. The presence of hyaluronidase andother lysosomal enzymes emphasizes that human trabecular cells arecapable of metabolizing hyaluronic acid and other extracellularmaterials. Potential mechanisms of trabecular cell damage in vitro maybe examined by evaluating, for example, the effects of extended passage,peroxide exposure, and laser treatment on cellular morphology.

[0116] Cell based assays based upon trabecular meshwork cells or othercell types can also be used to identify compounds which modulateexpression of an FRP gene, modulate translation of an FRP mRNA, or whichmodulate the stability of an FRP mRNA or protein. Accordingly, in oneembodiment, a cell which is capable of producing FRP, e.g., a trabecularmeshwork cell, is incubated with a test compound and the amount of FRPproduced in the cell medium is measured and compared to that producedfrom a cell which has not been contacted with the test compound. Thespecificity of the compound vis a vis FRP can be confirmed by variouscontrol analysis, e.g., measuring the expression of one or more controlgenes.

[0117] Compounds which can be tested include small molecules, proteins,and nucleic acids. In particular, this assay can be used to determinethe efficacity of FRP antisense molecules or ribozymes.

[0118] In another embodiment, the effect of a test compound ontranscription of an FRP gene is determined by transfection experimentsusing a reporter gene operatively linked to at least a portion of thepromoter of an FRP gene. A promoter region of a gene can be isolated,e.g., from a genomic library according to methods known in the art. Thereporter gene can be any gene encoding a protein which is readilyquantifiable, e.g., the luciferase or CAT gene, well known in the art.

[0119] In a preferred embodiment, the reporter gene is a natural orsynthetic gene which is transcriptionally activated in response to a Wntsignal. For example, the engrailed gene is activated in response to Wntinduction. Furthermore, increased expression of engrailed results in thetranscriptional induction of the hedgehog gene, which is therefor nowactivated in response to Wnt. Finally, synthetic reporter genes whichare activated by nuclear LEF(tcf)/beta-catenin also provide sensitivereporter genes for measuring Wnt induction.

[0120] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0121] 4.5. Methods of Treating Disease

[0122] A “glaucoma therapeutic,” whether an antagonist or agonist canbe, as appropriate, any of the preparations described above, includingisolated polypeptides, gene therapy constructs, antisense molecules,peptidomimetics, small molecules, non-nucleic acid, non-peptidic oragents identified in the drug assays provided herein.

[0123] The present invention provides for both prophylactic andtherapeutic methods of treating a subject having or likely to develop adisorder associated with aberrant FRP or Wnt pathway component genesexpression or activity, e.g., glaucoma.

[0124] 4.5.1. Prophylactic Methods

[0125] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant FRP or Wntpathway component genes expression or activity by administering to thesubject an agent which modulates FRP or Wnt pathway component genesexpression or at least one FRP or Wnt pathway component genes activity.Subjects at risk for such a disease can be identified by a diagnostic orprognostic assay, e.g., as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the FRP or Wnt pathway component genes aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of FRP or Wnt pathway componentgenes aberrancy, for example, a FRP or Wnt pathway component genesagonist or FRP or Wnt pathway component genes antagonist agent can beused for treating the subject prophylactically. The prophylactic methodsare similar to therapeutic methods of the present invention and arefurther discussed in the following subsections.

[0126] 4.5.2. Therapeutic Methods

[0127] In general, the invention provides methods for treating a diseaseor condition which is caused by or contributed to by an aberrant FRP orWnt pathway component genes activity comprising administering to thesubject an effective amount of a compound which is capable of modulatingan FRP or Wnt pathway component genes activity. Among the approacheswhich may be used to ameliorate disease symptoms involving an aberrantFRP or Wnt pathway component genes activity are, for example, antisense,ribozyme, and triple helix molecules or small organic agents asdescribed above. Examples of suitable compounds include the antagonists,agonists or homologues described in detail herein.

[0128] 4.5.3. Effective Dose

[0129] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining The LD₅₀ (The Dose Lethal To50% Of The Population) And The ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic induces arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0130] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include concentrations _(χ) the ED50 with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ to include concentrations_(χ) the IC₅₀ (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of symptoms) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

[0131] 4.5.4. Monitoring of Effects of FRP/Wnt Therapeutics DuringClinical Trials

[0132] The ability to target populations expected to show the highestclinical benefit, based on the FRP or Wnt pathway component genes ordisease genetic profile, can enable: 1) the repositioning of marketeddrugs with disappointing market results; 2) the rescue of drugcandidates whose clinical development has been discontinued as a resultof safety or efficacy limitations, which are patient subgroup-specific;and 3) an accelerated and less costly development for drug candidatesand more optimal drug labeling (e.g. since the use of FRP or Wnt pathwaycomponent genes as a marker is useful for optimizing effective dose).

[0133] The treatment of an individual with an FRP or Wnt pathwaycomponent genes therapeutic can be monitored by determining FRP or Wntpathway component genes characteristics, such as FRP or Wnt pathwaycomponent genes protein level or activity, FRP or Wnt pathway componentgenes mRNA level, and/or FRP or Wnt pathway component genestranscriptional level. This measurements will indicate whether thetreatment is effective or whether it should be adjusted or optimized.Thus, FRP or Wnt pathway component genes can be used as a marker for theefficacy of a drug during clinical trials.

[0134] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) comprising thesteps of (i) obtaining a preadministration sample from a subject priorto administration of the agent; (ii) detecting the level of expressionof an FRP or Wnt pathway component genes protein, mRNA, or genomic DNAin the preadministration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the FRP or Wnt pathway component genesprotein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the FRP or Wnt pathwaycomponent genes protein, mRNA, or genomic DNA in the preadministrationsample with the FRP or Wnt pathway component genes protein, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of FRP or Wnt pathway componentgenes to higher levels than detected, i.e., to increase theeffectiveness of the agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of FRP orWnt pathway component genes to lower levels than detected, i.e., todecrease the effectiveness of the agent.

[0135] Cells of a subject may also be obtained before and afteradministration of an FRP or Wnt pathway component genes therapeutic todetect the level of expression of genes other than FRP or Wnt pathwaycomponent genes, to verify that the FRP or Wnt pathway component genestherapeutic does not increase or decrease the expression of genes whichcould be deleterious. This can be done, e.g., by using the method oftranscriptional profiling. Thus, mRNA from cells exposed in vivo to anFRP or Wnt pathway component genes therapeutic and mRNA from the sametype of cells that were not exposed to the FRP or Wnt pathway componentgenes therapeutic could be reverse transcribed and hybridized to a chipcontaining DNA from numerous genes, to thereby compare the expression ofgenes in cells treated and not treated with an FRP or Wnt pathwaycomponent genes-therapeutic. If, for example an FRP or Wnt pathwaycomponent genes therapeutic turns on the expression of a proto-oncogenein an individual, use of this particular FRP or Wnt pathway componentgenes therapeutic may be undesirable.

[0136] 4.5.5. Formulation and Use

[0137] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or topical, oral,buccal, parenteral or rectal administration.

[0138] For such therapy, the compounds of the invention can beformulated for a variety of loads of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remmington's Pharmaceutical Sciences, MeadePublishing Co., Easton, Pa. Injection is not likely to be the preferredmethod of systemic administration; oral dosage forms are. Topicalophthalmic compositions the compounds of the invention can be formulatewith one or more pharmceutically acceptable excipients, such asbuffering agents, preservatives (including preservative adjuncts),tonicity-adjusting agents, surfactants, solubilizing agents stabilizingagents, comfort-enhancing agents, emollients, pH-adjusting agents andlubricants. Topically administrable ophthalmic compositions willgenerally be formulated at pH 4.5-8 and have an osmolarity of 26-320mOSm/kg. For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds of the invention can be formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the compounds may beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included.

[0139] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0140] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner. For administration by inhalation, thecompounds for use according to the present invention are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0141] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0142] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0143] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered topically, by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Other suitabledelivery systems include microspheres which offer the possibility oflocal noninvasive delivery of drugs over an extended period of time.This technology utilizes microspheres of precapillary size which can beinjected via a coronary catheter into any selected part of the e.g.heart or other organs without causing inflammation or ischemia. Theadministered therapeutic is slowly released from these microspheres andtaken up by surrounding tissue cells (e.g. endothelial cells).

[0144] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. in addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays or using suppositories. For topical administration, the oligomersof the invention are formulated into ointments, salves, gels, or creamsas generally known in the art. A wash solution can be used locally totreat an injury or inflammation to accelerate healing.

[0145] In clinical settings, a gene delivery system for the therapeuticFRP or Wnt pathway component gene can be introduced into a patient byany of a number of methods, each of which is familiar in the art. Forinstance, a pharmaceutical preparation of the gene delivery system canbe introduced by intraocular injection or systemically, e.g., byintravenous injection, and specific transduction of the protein in thetarget cells occurs predominantly from specificity of transfectionprovided by the gene delivery vehicle, cell-type or tissue-typeexpression due to the transcriptional regulatory sequences controllingexpression of the receptor gene, or a combination thereof. In otherembodiments, initial delivery of the recombinant gene is more limitedwith introduction into the animal being quite localized. For example,the gene delivery vehicle can be introduced by catheter (see U.S. Pat.No. 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994)PNAS 91: 3054-3057). An FRP or Wnt pathway component genes gene can bedelivered in a gene therapy construct by electroporation usingtechniques described, for example, by Dev et al. ((1994) Cancer TreatRev 20:105-115) or by transcleral iontophoresis.

[0146] The pharmaceutical preparation of the gene therapy construct orcompound of the invention can consist essentially of the gene deliverysystem in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle or compound is imbedded.Alternatively, where the complete gene delivery system can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

[0147] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0148] 4.6. Kits

[0149] The invention further provides kits for use in diagnostics orprognostic methods or for treating a disease or condition associatedwith an aberrant FRP or Wnt pathway component genes protein. Theinvention also provides kits for determining which FRP or Wnt pathwaycomponent genes therapeutic should be administered to a subject. Theinvention encompasses kits for detecting the presence of FRP or Wntpathway component genes mRNA or protein in a biological sample or fordetermining the presence of mutations or the identity of polymorphicregions in an FRP or Wnt pathway component genes gene. For example, thekit can comprise a labeled compound or agent capable of detecting FRP orWnt pathway component genes protein or mRNA in a biological sample;means for determining the amount of FRP or Wnt pathway component genesin the sample; and means for comparing the amount of FRP or Wnt pathwaycomponent genes in the sample with a standard. The compound or agent canbe packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect FRP or Wnt pathway componentgenes mRNA or protein.

[0150] In one embodiment, the kit comprises a pharmaceutical compositioncontaining an effective amount of an FRP or Wnt pathway component genesantagonist therapeutic and instruction for use in treating or preventinghypertension. In another embodiment, the kit comprises a pharmaceuticalcomposition comprising an effective amount of an FRP or Wnt pathwaycomponent genes agonist therapeutic and instructions for use in treatingeye disorders or diseases such as glaucoma. Generally, the kit comprisesa pharmaceutical composition comprising an effective amount of an FRP orWnt pathway component genes agonist or antagonist therapeutic andinstructions for use as a glaucoma therapeutic agent. For example, thekit can comprise a pharmaceutical composition comprising an effectiveamount of an FRP or Wnt pathway component genes agonist therapeutic andinstructions for use as an analgesic.

[0151] Yet other kits can be used to determine whether a subject has oris likely to develop a disease or condition associated with an aberrantFRP or Wnt pathway component genes activity. Such a kit can comprise,e.g., one or more nucleic acid probes capable of hybridizingspecifically to at least a portion of an FRP or Wnt pathway componentgenes gene or allelic variant thereof, or mutated form thereof.

[0152] 4.7. Additional Uses for FRP or Wnt Pathway Gene Proteins andNucleic Acids

[0153] The FRP or Wnt pathway component genes nucleic acids of theinvention can further be used in the following assays. In oneembodiment, the human FRP or Wnt pathway component genes nucleic acidhaving SEQ ID NO:1 or a portion thereof, or a nucleic acid whichhybridizes thereto can be used to determine the chromosomal localizationof an FRP or Wnt pathway component genes gene. Comparison of thechromosomal location of the FRP or Wnt pathway component genes gene withthe location of chromosomal regions which have been shown to beassociated with specific diseases or conditions, e.g., by linkageanalysis (coinheritance of physically adjacent genes), can be indicativeof diseases or conditions in which FRP or Wnt pathway component genesmay play a role. A list of chromosomal regions which have been linked tospecific diseases can be found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library) and at http://www3.ncbi.nlm.nih.gov/Omim/(OnlineMendelian Inheritance in Man). Furthermore, the FRP or Wnt pathwaycomponent genes gene can also be used as a chromosomal marker in geneticlinkage studies involving genes other than FRP or Wnt pathway componentgenes.

[0154] Chromosomal localization of a gene can be performed by severalmethods well known in the art. For example, Southern blot hybridizationor PCR mapping of somatic cell hybrids can be used for determining onwhich chromosome or chromosome fragment a specific gene is located.Other mapping strategies that can similarly be used to localize a geneto a chromosome or chromosomal region include in situ hybridization,prescreening with labeled flow-sorted chromosomes and preselection byhybridization to construct chromosome specific-cDNA libraries.

[0155] Furthermore, fluorescence in situ hybridization (FISH) of anucleic acid, e.g., an FRP or Wnt pathway component genes nucleic acid,to a metaphase chromosomal spread is a one step method that provides aprecise chromosomal location of the nucleic acid. This technique can beused with nucleic acids as short as 500 or 600 bases; however, cloneslarger than 2,000 bp have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Such techniques are described, e.g., in Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988). Using such techniques, a gene can be localized to a chromosomalregion containing from about 50 to about 500 genes.

[0156] If the FRP or Wnt pathway component genes gene is shown to belocalized in a chromosomal region which cosegregates, i.e., which isassociated, with a specific disease, the differences in the cDNA orgenomic sequence between affected and unaffected individuals aredetermined. The presence of a mutation in some or all of the affectedindividuals but not in any normal individuals, will be indicative thatthe mutation is likely to be causing or contributing to the disease.

[0157] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference. Thepractice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0158] 4.8. Pharmacogenomics

[0159] Knowledge of the particular alteration or alterations, resultingin defective or deficient FRP or Wnt pathway component genes or proteinsin an individual (the FRP or Wnt pathway component genes geneticprofile), alone or in conjunction with information on other geneticdefects contributing to the same disease (the genetic profile of theparticular disease) allows a customization of the therapy for aparticular disease to the individual's genetic profile, the goal of“pharmacogenomics”. For example, subjects having a specific allele of anFRP or Wnt pathway component genes gene may or may not exhibit symptomsof a particular disease or be predisposed of developing symptoms of aparticular disease. Further, if those subjects are symptomatic, they mayor may not respond to a certain drug, e.g., a specific FRP or Wntpathway component genes therapeutic, but may respond to another. Thus,generation of an FRP or Wnt pathway component genes genetic profile,(e.g., categorization of alterations in FRP or Wnt pathway componentgenes gene which are associated with the development of a particulardisease), from a population of subjects, who are symptomatic for adisease or condition that is caused by or contributed to by a defectiveand/or deficient FRP or Wnt pathway component genes gene and/or protein(an FRP or Wnt pathway component genes genetic population profile) andcomparison of an individual's FRP or Wnt pathway component genes profileto the population profile, permits the selection or design of drugs thatare expected to be safe and efficacious for a particular patient orpatient population (i.e., a group of patients having the same geneticalteration).

[0160] For example, an FRP or Wnt pathway component genes populationprofile can be performed, by determining the FRP or Wnt pathwaycomponent genes profile, e.g., the identity of FRP or Wnt pathwaycomponent genes genes, in a patient population having a disease, whichis caused by or contributed to by a defective or deficient FRP or Wntpathway component genes gene. Optionally, the FRP or Wnt pathwaycomponent genes population profile can further include informationrelating to the response of the population to an FRP or Wnt pathwaycomponent genes therapeutic, using any of a variety of methods,including, monitoring: 1) the severity of symptoms associated with theFRP or Wnt pathway component genes related disease, 2) FRP or Wntpathway component genes gene expression level, 3) FRP or Wnt pathwaycomponent genes mRNA level, and/or 4) FRP or Wnt pathway component genesprotein level. and (iii) dividing or categorizing the population basedon the particular genetic alteration or alterations present in its FRPor Wnt pathway component genes gene or an FRP or Wnt pathway componentgenes pathway gene. The FRP or Wnt pathway component genes geneticpopulation profile can also, optionally, indicate those particularalterations in which the patient was either responsive or non-responsiveto a particular therapeutic. This information or population profile, isthen useful for predicting which individuals should respond toparticular drugs, based on their individual FRP or Wnt pathway componentgenes profile.

[0161] In a preferred embodiment, the FRP or Wnt pathway component genesprofile is a transcriptional or expression level profile and step (i) iscomprised of determining the expression level of FRP or Wnt pathwaycomponent genes proteins, alone or in conjunction with the expressionlevel of other genes, known to contribute to the same disease. The FRPor Wnt pathway component genes profile can be measured in many patientsat various stages of the disease.

[0162] Pharmacogenomic studies can also be performed using transgenicanimals. For example, one can produce transgenic mice, e.g., asdescribed herein, which contain a specific allelic variant of an FRP orWnt pathway component genes gene. These mice can be created, e.g., byreplacing their wild-type FRP or Wnt pathway component genes gene withan allele of the human FRP or Wnt pathway component genes gene. Theresponse of these mice to specific FRP or Wnt pathway component genestherapeutics can then be determined.

[0163] 4.9. Transgenic Animals

[0164] The invention further provides for transgenic animals, which canbe used for a variety of purposes, e.g., to identify glaucomatherapeutics. Transgenic animals of the invention include non-humananimals containing mutations in nucleic acid sequences that result ininappropriately high levels of FRP (e.g. mutations in genes encodingtranscription factors that regulate expression of FRP). Alternatively,transgenic animals can contain mutations in Wnt signaling components,including: frizzled (Fz); disheveled (Dsh); glycogen synthase kinase 3(GSK3), protein kinase C (APC), β-catenins and high mobility group (HMG)proteins (e.g. LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor)). Suchanimals can be used, e.g., to determine the effect on phenotype ofinterfering with the expression in trabecular meshwork cells of geneswhose expression is regulated by Wnt signaling.

[0165] The transgenic animals can also be animals containing atransgene, such as reporter gene, under the control of an FRP promoteror fragment thereof. These animals are useful, e.g., for identifyingdrugs that modulate production of FRP, such as by modulating FRP geneexpression. An FRP gene promoter can be isolated, e.g., by screening ofa genomic library with an FRP cDNA fragment and characterized accordingto methods known in the art.

[0166] Yet other non-human animals within the scope of the inventioninclude genes encoding Wnt signaling components in which the expressionof the endogenous gene has been mutated or “knocked out”. These animalscould be useful to determine whether the absence of a Wnt signalingcomponent will result in a specific phenotype. Methods for obtainingtransgenic and knockout non-human animals are well known in the art andare discussed herein.

[0167] In a preferred embodiment, the invention provides transgenicnon-human animals for use in the development of glaucoma diagnostic andtherapeutic methods. For example, in certain preferred embodiments, thetransgenic animals of the invention comprise an heterologous FRPexpressing gene which results in an increase in the level of FRP geneexpression in an ocular tissue. In preferred embodiments, the oculartissue is the trabecular meshwork and the FRP-overexpressing cells aretrabecular meshwork cells. In still more preferred embodiments thetransgenic non-human animals expressing increased levels of FRP in thetrabecular meshwork cells have at least one symptom characteristic ofglaucoma, such as an increased intraocular pressure (IOP). In certainpreferred embodiments, the transgenic animals of the invention providean in vivo assay system for the screening of glaucoma therapeuticscompounds and the development of glaucoma diagnostics.

[0168] 4.9.1 Animal-based Systems

[0169] Another aspect of the present invention concerns transgenicanimals which are comprised of cells (of that animal) which contain atransgene of the present invention and which preferably (thoughoptionally) express an exogenous FRP protein in one or more cells in theanimal. A FRP transgene can encode the wild-type form of the protein, orcan encode homologs thereof, including both agonists and antagonists, aswell as antisense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of a FRP protein can be essential for many formsof lineage analysis and can additionally provide a means to assess theeffects of, for example, lack of FRP expression which might grosslyalter development in small patches of tissue within an otherwise normalembryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

[0170] Genetic techniques, which allow for the expression of transgenescan be regulated via site-specific genetic manipulation in vivo, areknown to those skilled in the art. For instance, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination of a target sequence. As usedherein, the phrase “target sequence” refers to a nucleotide sequencethat is genetically recombined by a recombinase. The target sequence isflanked by recombinase recognition sequences and is generally eitherexcised or inverted in cells expressing recombinase activity.Recombinase catalyzed recombination events can be designed such thatrecombination of the target sequence results in either the activation orrepression of expression of one of the subject FRP proteins. Forexample, excision of a target sequence which interferes with theexpression of a recombinant FRP gene, such as one which encodes anantagonistic homolog or an antisense transcript, can be designed toactivate expression of that gene. This interference with expression ofthe protein can result from a variety of mechanisms, such as spatialseparation of the FRP gene from the promoter element or an internal stopcodon. Moreover, the transgene can be made wherein the coding sequenceof the gene is flanked by recombinase recognition sequences and isinitially transfected into cells in a 3′ to 5′ orientation with respectto the promoter element. In such an instance, inversion of the targetsequence will reorient the subject gene by placing the 5′ end of thecoding sequence in an orientation with respect to the promoter elementwhich allow for promoter driven transcriptional activation.

[0171] The transgenic animals of the present invention all includewithin a plurality of their cells a transgene of the present invention,which transgene alters the phenotype of the “host cell” with respect toregulation of cell function, cell growth, death and/or differentiation.Since it is possible to produce transgenic organisms of the inventionutilizing one or more of the transgene constructs described herein, ageneral description will be given of the production of transgenicorganisms by referring generally to exogenous genetic material. Thisgeneral description can be adapted by those skilled in the art in orderto incorporate specific transgene sequences into organisms utilizing themethods and materials described below.

[0172] In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0173] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation expression of a recombinant FRP proteincan be regulated via control of recombinase expression.

[0174] Use of the cre/loxP recombinase system to regulate expression ofa recombinant FRP protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant FRP gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., a FRP gene and recombinase gene.

[0175] One advantage derived from initially constructing transgenicanimals containing a FRP transgene in a recombinase-mediated expressibleformat derives from the likelihood that the subject protein, whetheragonistic or antagonistic, can be deleterious upon expression in thetransgenic animal. In such an instance, a founder population, in whichthe subject transgene is silent in all tissues, can be propagated andmaintained. Individuals of this founder population can be crossed withanimals expressing the recombinase in, for example, one or more tissuesand/or a desired temporal pattern. Thus, the creation of a founderpopulation in which, for example, an antagonistic FRP transgene issilent will allow the study of progeny from that founder in whichdisruption of FRP mediated induction in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

[0176] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the FRP transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

[0177] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding thetrans-activating protein, e.g. a recombinase or a prokaryotic protein,is delivered to the tissue and caused to be expressed, such as in acell-type specific manner. By this method, a FRP A transgene couldremain silent into adulthood until “turned on” by the introduction ofthe trans-activator.

[0178] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2b, H-2d or H-2q haplotypes such as C57BL/6 or DBA/1. The line(s) usedto practice this invention may themselves be transgenics, and/or may beknockouts (i.e., obtained from animals which have one or more genespartially or completely suppressed).

[0179] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for micro-injection.In the mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

[0180] Normally, fertilized embryos are incubated in suitable mediauntil the pronuclei appear. At about this time, the nucleotide sequencecomprising the transgene is introduced into the female or malepronucleus as described below. In some species such as mice, the malepronucleus is preferred. It is most preferred that the exogenous geneticmaterial be added to the male DNA complement of the zygote prior to itsbeing processed by the ovum nucleus or the zygote female pronucleus. Itis thought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

[0181] Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

[0182] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0183] For the purposes of this invention a zygote is essentially theformation of a diploid cell which is capable of developing into acomplete organism. Generally, the zygote will be comprised of an eggcontaining a nucleus formed, either naturally or artificially, by thefusion of two haploid nuclei from a gamete or gametes. Thus, the gametenuclei must be ones which are naturally compatible, i.e., ones whichresult in a viable zygote capable of undergoing differentiation anddeveloping into a functioning organism. Generally, a euploid zygote ispreferred. If an aneuploid zygote is obtained, then the number ofchromosomes should not vary by more than one with respect to the euploidnumber of the organism from which either gamete originated.

[0184] In addition to similar biological considerations, physical onesalso govern the amount (e.g., volume) of exogenous genetic materialwhich can be added to the nucleus of the zygote or to the geneticmaterial which forms a part of the zygote nucleus. If no geneticmaterial is removed, then the amount of exogenous genetic material whichcan be added is limited by the amount which will be absorbed withoutbeing physically disruptive. Generally, the volume of exogenous geneticmaterial inserted will not exceed about 10 picoliters. The physicaleffects of addition must not be so great as to physically destroy theviability of the zygote. The biological limit of the number and varietyof DNA sequences will vary depending upon the particular zygote andfunctions of the exogenous genetic material and will be readily apparentto one skilled in the art, because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

[0185] The number of copies of the transgene constructs which are addedto the zygote is dependent upon the total amount of exogenous geneticmaterial added and will be the amount which enables the genetictransformation to occur. Theoretically only one copy is required;however, generally, numerous copies are utilized, for example,1,000-20,000 copies of the transgene construct, in order to insure thatone copy is functional. As regards the present invention, there willoften be an advantage to having more than one functioning copy of eachof the inserted exogenous DNA sequences to enhance the phenotypicexpression of the exogenous DNA sequences.

[0186] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0187] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0188] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by PCR, Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0189] Alternative or additional methods for evaluating the presence ofthe transgene include, without limitation, suitable biochemical assayssuch as enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

[0190] Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

[0191] The transgenic animals produced in accordance with the presentinvention will include exogenous genetic material. As set out above, theexogenous genetic material will, in certain embodiments, be a DNAsequence which results in the production of a FRP protein (eitheragonistic or antagonistic), and antisense transcript, or a FRP mutant.Further, in such embodiments the sequence may be attached to atranscriptional control element, e.g., a promoter, which preferablyallows the expression of the transgene product in a specific type ofcell.

[0192] Retroviral infection can also be used to introduce transgene intoa non-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0193] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

[0194] In one embodiment, gene targeting, which is a method of usinghomologous recombination to modify an animal's genome, can be used tointroduce changes into cultured embryonic stem cells. By targeting a FRPgene of interest in ES cells, these changes can be introduced into thegermlines of animals to generate chimeras. The gene targeting procedureis accomplished by introducing into tissue culture cells a DNA targetingconstruct that includes a segment homologous to a target FRP locus, andwhich also includes an intended sequence modification to the FRP genomicsequence (e.g., insertion, deletion, point mutation). The treated cellsare then screened for accurate targeting to identify and isolate thosewhich have been properly targeted.

[0195] Gene targeting in embryonic stem cells is in fact a schemecontemplated by the present invention as a means for disrupting a FRPgene function through the use of a targeting transgene constructdesigned to undergo homologous recombination with one or more FRPgenomic sequences. The targeting construct can be arranged so that, uponrecombination with an element of a FRP gene, a positive selection markeris inserted into (or replaces) coding sequences of the gene. Theinserted sequence functionally disrupts the FRP gene, while alsoproviding a positive selection trait. Exemplary FRP targeting constructsare described in more detail below.

[0196] Generally, the embryonic stem cells (ES cells ) used to producethe knockout animals will be of the same species as the knockout animalto be generated. Thus for example, mouse embryonic stem cells willusually be used for generation of knockout mice.

[0197] Embryonic stem cells are generated and maintained using methodswell known to the skilled artisan such as those described by Doetschmanet al. (1985) J. Embryol. Exp. Morphol. 87:27-45). Any line of ES cellscan be used, however, the line chosen is typically selected for theability of the cells to integrate into and become part of the germ lineof a developing embryo so as to create germ line transmission of theknockout construct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934) Still another preferred ES cell line is the WW6cell line (Ioffe et al. (1995) PNAS 92:7357-7361). The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth by Robertsonin: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]).

[0198] Insertion of the knockout construct into the ES cells can beaccomplished using a variety of methods well known in the art includingfor example, electroporation, microinjection, and calcium phosphatetreatment. A preferred method of insertion is electroporation.

[0199] Each knockout construct to be inserted into the cell must firstbe in the linear form. Therefore, if the knockout construct has beeninserted into a vector (described infra), linearization is accomplishedby digesting the DNA with a suitable restriction endonuclease selectedto cut only within the vector sequence and not within the knockoutconstruct sequence.

[0200] For insertion, the knockout construct is added to the ES cellsunder appropriate conditions for the insertion method chosen, as isknown to the skilled artisan. Where more than one construct is to beintroduced into the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

[0201] If the ES cells are to be electroporated, the ES cells andknockout construct DNA are exposed to an electric pulse using anelectroporation machine and following the manufacturer's guidelines foruse. After electroporation, the ES cells are typically allowed torecover under suitable incubation conditions. The cells are thenscreened for the presence of the knockout construct.

[0202] Screening can be accomplished using a variety of methods. Wherethe marker gene is an antibiotic resistance gene, for example, the EScells may be cultured in the presence of an otherwise lethalconcentration of antibiotic. Those ES cells that survive have presumablyintegrated the knockout construct. If the marker gene is other than anantibiotic resistance gene, a Southern blot of the ES cell genomic DNAcan be probed with a sequence of DNA designed to hybridize only to themarker sequence Alternatively, PCR can be used. Finally, if the markergene is a gene that encodes an enzyme whose activity can be detected(e.g., b-galactosidase), the enzyme substrate can be added to the cellsunder suitable conditions, and the enzymatic activity can be analyzed.One skilled in the art will be familiar with other useful markers andthe means for detecting their presence in a given cell. All such markersare contemplated as being included within the scope of the teaching ofthis invention.

[0203] The knockout construct may integrate into several locations inthe ES cell genome, and may integrate into a different location in eachES cell's genome due to the occurrence of random insertion events. Thedesired location of insertion is in a complementary position to the DNAsequence to be knocked out, e.g., the FRP coding sequence,transcriptional regulatory sequence, etc. Typically, less than about1-5% of the ES cells that take up the knockout construct will actuallyintegrate the knockout construct in the desired location. To identifythose ES cells with proper integration of the knockout construct, totalDNA can be extracted from the ES cells using standard methods. The DNAcan then be probed on a Southern blot with a probe or probes designed tohybridize in a specific pattern to genomic DNA digested with particularrestriction enzyme(s). Alternatively, or additionally, the genomic DNAcan be amplified by PCR with probes specifically designed to amplify DNAfragments of a particular size and sequence (i.e., only those cellscontaining the knockout construct in the proper position will generateDNA fragments of the proper size).

[0204] After suitable ES cells containing the knockout construct in theproper location have been identified, the cells can be inserted into anembryo. Insertion may be accomplished in a variety of ways known to theskilled artisan, however a preferred method is by microinjection. Formicroinjection, about 10-30 cells are collected into a micropipet andinjected into embryos that are at the proper stage of development topermit integration of the foreign ES cell containing the knockoutconstruct into the developing embryo. For instance, as the appendedExamples describe, the transformed ES cells can be microinjected intoblastocytes.

[0205] The suitable stage of development for the embryo used forinsertion of ES cells is very species dependent, however for mice it isabout 3.5 days. The embryos are obtained by perfusing the uterus ofpregnant females. Suitable methods for accomplishing this are known tothe skilled artisan, and are set forth by, e.g., Bradley et al. (supra).

[0206] While any embryo of the right stage of development is suitablefor use, preferred embryos are male. In mice, the preferred embryos alsohave genes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo). Thus, for example, if the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

[0207] After the ES cell has been introduced into the embryo, the embryomay be implanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

[0208] Offspring that are born to the foster mother may be screenedinitially for mosaic coat color where the coat color selection strategy(as described above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by Southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross, as well as mice that are known heterozygotes andwild type mice.

[0209] Other means of identifying and characterizing the knockoutoffspring are available. For example, Northern blots can be used toprobe the mRNA for the presence or absence of transcripts encodingeither the gene knocked out, the marker gene, or both. In addition,Western blots can be used to assess the level of expression of the FRPgene knocked out in various tissues of the offspring by probing theWestern blot with an antibody against the particular FRP protein, or anantibody against the marker gene product, where this gene is expressed.Finally, in situ analysis (such as fixing the cells and labeling withantibody) and/or FACS (fluorescence activated cell sorting) analysis ofvarious cells from the offspring can be conducted using suitableantibodies to look for the presence or absence of the knockout constructgene product.

[0210] Yet other methods of making knock-out or disruption transgenicanimals are also generally known. See, for example, Manipulating theMouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g.by homologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a FRP-gene can becontrolled by recombinase sequences (described infra).

[0211] Animals containing more than one knockout construct and/or morethan one transgene expression construct are prepared in any of severalways. The preferred manner of preparation is to generate a series ofmammals, each containing one of the desired transgenic phenotypes. Suchanimals are bred together through a series of crosses, backcrosses andselections, to ultimately generate a single animal containing alldesired knockout constructs and/or expression constructs, where theanimal is otherwise congenic (genetically identical) to the wild typeexcept for the presence of the knockout construct(s) and/ortransgene(s).

[0212] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0213] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

[0214] 5. 1 Association of Frizzle Related Protein 1 (FRP-1) andGlaucoma

[0215] Materials and Methods

[0216] Frizzled Related Protein cDNA Sequence Identified by RNADifferential Display     AACAGCCTGCCTGTCCCCCCGCACTTTTTACATATATTTGTTTCATTTCTGCAGATGGAAAGTTGACATGGGTGGGGTGTCCCCATCCAGCGAGAGAGTTTCAAAAGCAAAACATCTCTGCAGTTTTTCCCAAGTACCCTGAGATACTTCCCAAAGCCGTTATGTTTAATCAGCGATGTATATAAGCCAGTTCACTTAGACAACTTTACCCTTCTTGTCCAATGTACAGGAAGTAGTTCT

[0217] 5.2. Expression of Recombinant FRP or Wnt Pathway Genes in COSCells

[0218] This example describes a method for producing recombinant fulllength human FRP or Wnt pathway component genes in a mammalianexpression system.

[0219] An expression construct containing a nucleic acid encoding a fulllength human FRP or Wnt pathway component genes protein, or a solubleFRP or Wnt pathway component genes protein can be constructed asfollows. A nucleic acid encoding the full length human FRP or Wntpathway component genes protein or a soluble form of FRP or Wnt pathwaycomponent genes protein described above is obtained by reversetranscription (RT-PCR) of mRNA extracted from human cells expressing FRPor Wnt pathway component genes, e.g., human trabecullar meshwork cellsusing PCR primers based on the sequence set forth in SEQ ID NO: 1. ThePCR primers further contain appropriate restriction sites forintroduction into the expression plasmid. The amplified nucleic acid isthen inserted in a eukaryotic expression plasmid such as pcDNAI/Amp (InVitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gens, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the full length human FRP or Wnt pathway componentgenes and a HA or myc tag fused in frame to its 3′ end is then clonedinto the polylinker region of the. The HA tag corresponds to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to FRP or Wntpathway component genes allows easy detection of the recombinant proteinwith an antibody that recognizes the HA epitope.

[0220] For expression of the recombinant FRP or Wnt pathway componentgenes, COS cells are transfected with the expression vector byDEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the FRP or Wnt pathway component genes-HA protein can bedetected by radiolabelling and immunoprecipitation with an anti-HAantibody. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, (1988)). For this, transfected cells arelabeled with ³⁵S-cysteine two days post transfection. The cells, oralternatively the culture media (e.g., for the soluble FRP or Wntpathway component genes) is then collected and the FRP or Wnt pathwaycomponent genes protein immunoprecipitated with an HA specificmonoclonal antibody. Alternatively, expression of the recombinantprotein can be detected by Western blot analysis. To determine whetherfull length FRP or Wnt pathway component genes is a membrane protein,and/or a secreted protein, the cells transfected with a vector encodingthe fall length FRP or Wnt pathway component genes protein can be lysedwith detergent (RIPA buffer (1 50 mM NaCl 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)).Proteins precipitated can then be analyzed on SDS-PAGE gel. Thus, thepresence of FRP or Wnt pathway component genes in the cell will beindicative that the full length FRP or Wnt pathway component genes canbe membrane bound and the presence of FRP or Wnt pathway component genesin the supernatant will be indicative that the protein can also be in asoluble form, whether produced as a secreted protein or released byleakage from the cell.

[0221] Equivalents

[0222] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents of thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A method for diagnosing glaucoma comprising detecting anaberrant level or bioactivity of a Wnt pathway component or a frizzledrelated protein gene product in a patient sample.
 2. The method of claim1, wherein the patient sample comprises cells of the trabecular meshworkcells tissue or patient tears.
 3. The method of claim 1, wherein anaberrantly high level of a frizzled related protein gene product isdiagnostic of a glaucomatous state.
 4. The method of claim 3 wherein thefrizzled related protein gene product is an FRP-1 nucleic acid or anFRP-1 polypeptide.
 5. The method of claim 1, wherein the Wnt pathwaycomponent is selected from the group consisting of: a Wnt gene, aFrizzled gene, a glycogen synthase-kinase gene, a protein kinase C gene,a beta-catenin gene, a TCF gene, a TCF regulated gene and a hedgehoggene.
 6. The method of claim 1, wherein the Wnt pathway componentbioactivity is a beta-catenin bioactivity.
 7. The method of claim 6,wherein the beta-catenin bioactivity is measured by determining thelevel of phosphorylated beta-catenin.
 8. The method of claim 6, whereinan aberrantly level of phosphorylated beta-catenin is diagnostic ofglaucoma.
 9. The method of claim 1, wherein the Wnt pathway componentbioactivity is a kinase.
 10. The method of claim 8, wherein the kinaseis a glycogen synthase kinase-3 or a protein kinase C.
 11. The method ofclaim 10, wherein an aberrantly level of a glycogen synthase kinase-3activity or a protein kinase C activity is diagnostic of a glaucomatousstate.
 12. A method for diagnosing glaucoma comprising detecting atleast one human polymorphic allele in a Wnt pathway component encodinggene or in a frizzled related protein encoding gene obtained from apatient sample.
 13. The method of claim 12, wherein the patient sampleis a blood sample or a cheek swab sample.
 14. The method of claim 12,wherein the human polymorphic allele is in the FRP-1 gene.
 15. Themethod of claim 14, wherein the human polymorphic allele is in the FRP-1gene promoter.
 16. The method of claim 15, FRP-1 gene promoterpolymorphic allele is associated with increased expression of an FRP-1gene product.
 17. A method of identifying an anti-glaucomatous compoundcomprising: contacting a cell expressing a Wnt pathway component or afrizzled related protein gene product with a test compound; anddetecting a level or bioactivity of said Wnt pathway component or saidfrizzled related protein gene product in the presence of the testcompound; wherein an increase or decrease in the level or bioactivity ofthe Wnt pathway component or the frizzled related protein gene productin the presence of the test compound as compared to the level orbioactivity detected in the absence of the test compound identifies saidtest compound as an anti-glaucomatous compound.
 18. The method of claim17, wherein a decrease in the level or bioactivity of a frizzled relatedprotein gene product detected in the presence of the test as compared tothe level or bioactivity detected in the absence of the test compoundidentifies said test compound as an anti-glaucomatous compound.
 19. Themethod of claim 18, wherein the frizzled related protein gene is FRP-1.20. The method of claim 17, wherein and increase in the level orbioactivity of a Wnt pathway component detected in the presence of thetest as compared to the level or bioactivity detected in the absence ofthe test compound identifies said test compound as an anti-glaucomatouscompound.
 21. The method of claim 20, wherein the Wnt pathway componentis selected from the group consisting of: a Wnt gene product, a Frizzledgene product, a glycogen synthase-kinase gene product, a protein kinaseC gene product, a beta-catenin gene product, a TCF gene product, a TCFregulated gene product, and a hedgehog gene product.
 22. The method ofclaim 20, wherein the Wnt pathway component bioactivity is abeta-catenin bioactivity.
 23. The method of claim 22, wherein thebeta-catenin bioactivity is measured by determining the level ofphosphorylated beta-catenin.
 24. The method of claim 23, wherein adecrease in the level of phosphorylated beta-catenin in the presence ofthe test compound as compared to the level of phosphorylatedbeta-catenin in the absence of the test compound identifies said testcompound as an anti-glaucomatous compound.
 25. The method of claim 17,wherein the Wnt pathway component bioactivity is a kinase activity. 26.The method of claim 25, wherein the kinase activity is a glycogensynthase kinase-3 activity and wherein a change in the level of thekinase activity in the presence of the test compound as compared to thelevel of the kinase activity in the absence of the test compoundidentifies said test compound as an anti-glaucomatous compound.
 27. Themethod of claim 25, wherein the kinase activity is a protein kinase Cactivity and wherein a change in the level of the kinase activity in thepresence of the test compound as compared to the level of the kinaseactivity in the absence of the test compound identifies said testcompound as an anti-glaucomatous compound.
 28. A method of identifyingan anti-glaucomatous compound comprising: contacting a cell sample witha test compound; and measuring a level or bioactivity of a Wntresponsive gene; wherein an increase in the level of the Wnt responsivegene in the presence of the test compound as compared to the level orbioactivity of the Wnt responsive gene in the absence of the testcompound identifies said test compound as an anti-glaucomatous compound.29. The method of claim 28, wherein the Wnt responsive gene is selectedfrom the group consisting of: a hedgehog gene, an engrailed gene, aLef/tcf-regulated gene, a synthetic reporter gene.
 30. A method fortreating glaucoma in a human subject, comprising administering to thesubject, a therapeutically effective amount of a compound that modulatesthe level or bioactivity of a Wnt pathway component or frizzled relatedprotein gene product.
 31. The method of claim 24, wherein the compoundis selected from the group consisting of: a protein, peptide,peptidomimetic, small molecule or nucleic acid.
 32. The method of claim31, wherein the nucleic acid is selected from the group consisting of: agene, antisense, ribozyme and triplex nucleic acid.
 33. The method ofclaim 31, wherein the nucleic acid is a frizzled related protein genenucleic acid.
 34. The method of claim 30, wherein the compound is anantagonist of a frizzled related protein gene product.
 35. The method ofclaim 34, wherein the compound is a gene therapeutic.
 36. The method ofclaim 34, wherein the compound is a protein therapeutic.
 37. The methodof claim 30, wherein the compound is an agent that increases the levelof a Wnt pathway component gene or bioactivity.
 38. A method of claim30,;wherein the compound is comprised of an antagonist of a mutantfrizzled related protein gene or gene product.
 39. A method of claim 38,wherein the compound is an antisense, ribozyme or triple helix molecule.40. A method of claim 38, wherein the compound is a small molecule,peptide, or eptidomimetic.
 41. A method of claim 38, wherein thecompound is an antibody.
 42. A method for screening for a frizzledrelated protein agonist or antagonist comprising the steps of: a)combining a frizzled related protein polypeptide or bioactive fragmentsthereof, a frizzled related protein binding partner and a test compoundunder conditions wherein, but for the test compound, the frizzledrelated protein protein and frizzled related protein binding partnerbinding partner are able to interact; and b) detecting the extent towhich a frizzled related protein/frizzled related protein bindingpartner complex is formed in the presence of the test compound, whereinan increased amount of complex formation in the presence of the testcompound relative to in the absence of a test compound indicates thatthe test compound is a frizzled related protein agonist and a decreasedamount of complex formation in the presence of the test compoundrelative to in the absence of the test compound indicates that the testcompound is a frizzled related protein antagonist.
 43. A method of claim43, which additionally comprises the step of preparing a pharmaceuticalcomposition from the test compound.